Streptomyces phage ?C31 integrase (Int)-a large serine site-specific recombinase-is autonomous for phage integration (attP x attB recombination) but is dependent on the phage coded gp3, a recombination directionality factor (RDF), for prophage excision (attL x attR recombination). A previously described activating mutation, E449K, induces Int to perform attL x attR recombination in the absence of gp3, albeit with lower efficiency. E449K has no adverse effect on the competence of Int for attP x attB recombination. Int(E449K) resembles Int in gp3 mediated stimulation of attL x attR recombination and inhibition of attP x attB recombination. Using single-molecule analyses, we examined the mechanism by which E449K activates Int for gp3-independent attL x attR recombination. The contribution of E449K is both thermodynamic and kinetic. First, the mutation modulates the relative abundance of Int bound attL-attR site complexes, favoring pre-synaptic (PS) complexes over non-productively bound complexes. Roughly half of the synaptic complexes formed from Int(E449K) pre-synaptic complexes are recombination competent. By contrast, Int yields only inactive synapses. Second, E449K accelerates the dissociation of non-productively bound complexes and inactive synaptic complexes formed by Int. The extra opportunities afforded to Int(E499K) in reattempting synapse formation enhances the probability of success at fruitful synapsis.Background To date, no report on COVID-19 pediatric patients in a large urban center with data on underlying comorbidities and co-infection for hospitalized cases has been published. Methods Case series of Chicago COVID-19 patients aged 0-17 years reported to Chicago Department of Public Health (CDPH) from 3/5/20-4/8/20. Enhanced case investigation performed. Chi-square and Wilcoxon two-sample tests to compare characteristics among hospitalized and non-hospitalized cases. Results During March 5-April 8, 2020, 6369 lab-confirmed cases of COVID-19 were reported to CDPH; 64 (1.0%) were among children 0-17 years. Ten patients (16%) were hospitalized, seven (70%) required intensive care (ICU); median length of hospitalization 4 days (range 1-14). Reported fever and dyspnea were significantly higher in hospitalized patients compared to non-hospitalized patients (9/10 vs. 28/54, p = 0.04 and 7/10 vs. 10/54, p = 0.002, respectively). Hospitalized patients were significantly younger than non-hospitalized patients (median, 3.5 years vs. 12 years; p = 0.03) and all either had an underlying comorbidity or co-infection. Among the 34 unique households with multiple laboratory-confirmed infections, median number of laboratory-confirmed infections was 2 (range 2-5), and 31 (91%) households had at least one COVID-19 infected adult. For 15 households with available data to assess transmission, 11 (73%) were adult-to-child, 2 (13%) child-to-child, and 2 (13%) child-to-adult. Conclusions Enhanced case investigation of hospitalized patients revealed that underlying comorbidities and co-infection might have contributed to severe disease. Given frequency of household transmission, healthcare providers should consider alternative dispositional planning for affected families of children living with comorbidities.The Society for Neuro-Oncology (SNO) has long recognized that strategic investments back into the field pay significant dividends in terms of growing and broadening the reach of the organization and supporting the academic careers of our members. Over the years, SNO has created a number of committees, each tasked with addressing an unmet need in the field or to increase the participation and presence of an underrepresented group. This installment of the series of articles commemorating the 25th Anniversary of the Society for Neuro-Oncology will focus on the work of the International Outreach Committee, the Young Investigators Committee, the Wellness Committee, and the recently established Women and Diversity Committee.The systematic perturbation of genomes using CRISPR/Cas9 deciphers gene function at an unprecedented rate, depth and ease. Commercially available sgRNA libraries typically contain tens of thousands of pre-defined constructs, resulting in a complexity challenging to handle. In contrast, custom sgRNA libraries comprise gene sets of self-defined content and size, facilitating experiments under complex conditions such as in vivo systems. To streamline and upscale cloning of custom libraries, we present CLUE, a bioinformatic and wet-lab pipeline for the multiplexed generation of pooled sgRNA libraries. CLUE starts from lists of genes or pasted sequences provided by the user and designs a single synthetic oligonucleotide pool containing various libraries. At the core of the approach, a barcoding strategy for unique primer binding sites allows amplifying different user-defined libraries from one single oligonucleotide pool. We prove the approach to be straightforward, versatile and specific, yielding uniform sgRNA distributions in all resulting libraries, virtually devoid of cross-contaminations. For in silico library multiplexing and design, we established an easy-to-use online platform at www.crispr-clue.de. All in all, CLUE represents a resource-saving approach to produce numerous high quality custom sgRNA libraries in parallel, which will foster their broad use across molecular biosciences.The nucleolus is a membrane-less nuclear structure that disassembles when cells undergo mitosis. During mitosis, nucleolar factors are thus released from the nucleolus and dynamically change their subcellular localization; however, their functions remain largely uncharacterised. Here, we found that a nucleolar factor called nucleolar protein 11 (NOL11) forms a protein complex with two tryptophan-aspartic acid (WD) repeat proteins named WD-repeat protein 43 (WDR43) and Cirhin in mitotic cells. This complex, referred to here as the NWC (NOL11-WDR43-Cirhin) complex, exists in nucleoli during interphase and translocates to the periphery of mitotic chromosomes, i.e., perichromosomal regions. https://www.selleckchem.com/products/oligomycin-a.html During mitotic progression, both the congression of chromosomes to the metaphase plate and sister chromatid cohesion are impaired in the absence of the NWC complex, as it is required for the centromeric enrichment of Aurora B and the associating phosphorylation of histone H3 at threonine 3. These results reveal the characteristics of a novel protein complex consisting of nucleolar proteins, which is required for regulating kinetochores and centromeres to ensure faithful chromosome segregation.